The transaminase enzymes aspartate 2-oxoglutarate aminotransferase, hereafter abbreviated AST, and alanine 2-oxoglutarate aminotransferase, hereafter abbreviated ALT, are found in normal human serum in both the apo (inactive) and holo (active) forms. The holo or active form of the transaminase enzymes contain the coenzyme factor pyridoxal phosphate, hereafter called PLP. The apo or inactive form of the enzymes lack PLP. The holo form of AST catalyzes the transamination between L-aspartate and 2-oxoglutarate to form oxalacetate and glutamate. Similarly, the holo form of ALT catalyzes the transamination between L-alanine and 2-oxoglutarate to form pyruvate and glutamate. Analysis of AST activity in serum is commonly used in the diagnosis of human diseases or disorders such as myocardial infarction, and liver disorders such as toxic hepatitis, cirrhosis, and obstructive jaundice, infectious mononulceosis, acute muscular dystrophy, and acute pancreatitis. Analysis of ALT activity in human serum is used in the diagnosis of liver disease including those mentioned above with the ratio of ALT/AST being employed in further confirmation of liver dysfunction.
An assay for AST was developed by Karmen, J. Clin. Invest. 34, 131 (1955). This method, employing the measurement of oxalacetate using the secondary reaction of malic dehydrogenase and NADH, was modified by Henry, et al., Am. J. Clin. Path, 34, 381 (1960), and Amador and Wacker, Clin. Chem., 8, 343 (1962). Additional modifications including utilization of lactate dehydrogenase to rid the total reaction mixture of endogenous serum pyruvate and the selection of optimum concentrations of aspartate and 2-oxoglutarate for measurement of the isoenzymes of AST were recommended by the German Society of Clinical Chemistry, Z. Klin. Chem. Klin. Biochem. 8, 659 (1970). Most recently, measurement of total AST rather than just the holoenzyme constituency has prompted the utilization by Rej and Vanderline of pyridoxal phosphate in a reaction mixture devoid of phosphate, but including organic buffers such as imidazole, diethenolamine, or tris (hydroxymethyl) methyl aminopropane sulfonic acid (TAPS), Clin. Chem. 21, 1585 (1975).
The key reactions may be summarized as follows: ##STR1## The measurement of the rate of decrease in NADH concentration as measured at 340 nm is proportional to the AST activity once the endogenous pyruvate has been eliminated by the lactate dehydrogenase reaction.
Likewise, an assay for ALT was developed by Karmen et al., J. Clin. Invest. 34, 126 (1955), employing the measurement of pyruvate using the secondary reaction of lactate dehydrogenase and NADH. Improvements involving selection of optimum concentrations of alanine and 2-oxoglutarate as recommended by the German Society of Clinical Chemistry, Z. Klin. Chem. Klin. Biochem. 10, 182 (1972), and inclusion of PLP for measurement of total ALT, Jung & Egger, Clin. Chim. Acta, 64, 329 (1975) have been made. The key reactions may be summarized as follows: ##STR2## As in the AST activity determination, the measurement of the rate of decrease in NADH concentration as measured in the ultraviolet is proportional to the ALT activity once endogenous serum pyruvate has been eliminated.